CX3CR1 defines functionally distinct intestinal mononuclear phagocyte subsets which maintain their respective functions during homeostatic and inflammatory conditions

Intestinal mononuclear phagocytes (iMNP) are critically involved in mucosal immunity and tissue homeostasis. Two major non-overlapping populations of iMNP have been identified in mice. CD103(+) iMNP represent a migratory population capable of inducing tolerogenic responses, whereas CX3CR1(+) iMNP are resident cells with disease-promoting potential. CX3CR1(+) iMNP can further be subdivided based on differential expression of CX3CR1. Using CX3CR1(GFP/+) ×RAG2(-/-) mice, we demonstrate that CX3CR1(hi) and CX3CR1(lo) iMNP clearly differ with respect to their morphological and functional properties. Compared with CX3CR1(hi) iMNP, CX3CR1(lo) iMNP are polarised towards pro-inflammatory responses already under homeostatic conditions. During a CD4(+) T-cell-induced colitis, CX3CR1(lo) cells accumulate in the inflamed mucosa and upregulate the expression of pro-inflammatory cytokines and triggering receptor expressed on myeloid cells-1 (TREM-1). In contrast, CX3CR1(hi) iMNP retain their non-inflammatory profile even during intestinal inflammation. These findings identify two functionally distinct iMNP subsets based on differential expression of CX3CR1 and indicate an unanticipated stability of iMNP.

Role of meprins to protect ileal mucosa of Crohn's disease patients from colonization by adherent-invasive E. coli

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to adhere to and invade intestinal epithelial cells (IEC), and to survive within macrophages. The interaction of AIEC with IEC depends on bacterial factors mainly type 1 pili, flagella, and outer membrane proteins. In humans, proteases can act as host defence mechanisms to counteract bacterial colonization. The protease meprin, composed of multimeric complexes of the two subunits alpha and beta, is abundantly expressed in IECs. Decreased levels of this protease correlate with the severity of the inflammation in patients with inflammatory bowel disease. The aim of the present study was to analyze the ability of meprin to modulate the interaction of AIEC with IECs. In patients with ileal CD we observed decreased levels of meprins, in particular that of meprin β. Dose-dependent inhibition of the abilities of AIEC strain LF82 to adhere to and invade intestinal epithelial T84 cells was observed when bacteria were pre-treated with both exogenous meprin α and meprin β. Dose-dependent proteolytic degradation of type 1 pili was observed in the presence of active meprins, but not with heat-inactivated meprins, and pretreatment of AIEC bacteria with meprins impaired their ability to bind mannosylated host receptors and led to decreased secretion of the pro-inflammatory cytokine IL-8 by infected T84 cells. Thus, decreased levels of protective meprins as observed in CD patients may contribute to increased AIEC colonization.

Eosinophils in the gastrointestinal tract: friends or foes?

Eosinophils play an important role in the mucosal immune system of the gastrointestinal tract under resting and under inflammatory conditions. Under steady-state conditions, the mucosa of the digestive tract is the only organ harboring a substantial number of eosinophils, which, if need be, get activated and exert several effector and immunoregulatory functions. The precise function of these late-phase inflammatory cells is not yet completely understood. Nevertheless, it has recently been demonstrated that lipopolysaccharides from gram-negative bacteria activate eosinophils to rapidly release mitochondrial DNA in the extracellular space. Released mitochondrial DNA and eosinophil granule proteins form extracellular structures able to bind and inactivate bacteria. These findings suggest a novel mechanism of eosinophil-mediated innate immune responses that might be important in maintaining the intestinal barrier function. Moreover, eosinophils also play a crucial role in several inflammatory conditions, such as intestinal infections, immune-mediated inflammations and hypersensitivity reactions. Under chronic inflammatory conditions, the ability of the eosinophils to induce repair can lead to pathological sequelae in the tissue, such as esophageal remodeling in eosinophilic esophagitis. It is established that the uncontrolled eosinophilic inflammation induces fibrosis, esophageal wall thickening and strictures leading to damage that results in a loss of esophageal function. One potential mechanism of this remodeling is so-called 'epithelial mesenchymal transition', which is triggered by eosinophils and is potentially reversible under successful anti-eosinophil treatment. Therefore, eosinophils may act either as friends or as foes, depending on the microenvironment.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.

Adaptations of intestinal macrophages to an antigen-rich environment

Intestinal macrophages, preferentially located in the subepithelial lamina propria, represent in humans the largest pool of tissue macrophages. To comply with their main task, i.e. the efficient removal of microbes and particulate matter that might have gained access to the mucosa from the intestinal lumen while maintaining local tissue homeostasis, several phenotypic and functional adaptations evolved. Most notably, microbe-associated molecular pattern (MAMP) receptors, including the lipopolysaccharide receptors CD14 and TLR4, but also the Fc receptors for IgA and IgG are absent on most intestinal Mø. Here we review recent findings on the phenotypic and functional adaptations of intestinal Mø and their implications for the pathogenesis of inflammatory bowel diseases.

Contribution of mucosal maltase-glucoamylase activities to mouse small intestinal starch alpha-glucogenesis

Digestion of starch requires activities provided by 6 interactive small intestinal enzymes. Two of these are luminal endo-glucosidases named alpha-amylases. Four are exo-glucosidases bound to the luminal surface of enterocytes. These mucosal activities were identified as 4 different maltases. Two maltase activities were associated with sucrase-isomaltase. Two remaining maltases, lacking other identifying activities, were named maltase-glucoamylase. These 4 activities are better described as alpha-glucosidases because they digest all linear starch oligosaccharides to glucose. Because confusion persists about the relative roles of these 6 enzymes, we ablated maltase-glucoamylase gene expression by homologous recombination in Sv/129 mice. We assayed the alpha-glucogenic activities of the jejunal mucosa with and without added recombinant pancreatic alpha-amylase, using a range of food starch substrates. Compared with wild-type mucosa, null mucosa or alpha-amylase alone had little alpha-glucogenic activity. alpha-Amylase amplified wild-type and null mucosal alpha-glucogenesis. alpha-Amylase amplification was most potent against amylose and model resistant starches but was inactive against its final product limit-dextrin and its constituent glucosides. Both sucrase-isomaltase and maltase-glucoamylase were active with limit-dextrin substrate. These mucosal assays were corroborated by a 13C-limit-dextrin breath test. In conclusion, the global effect of maltase-glucoamylase ablation was a slowing of rates of mucosal alpha-glucogenesis. Maltase-glucoamylase determined rates of digestion of starch in normal mice and alpha-amylase served as an amplifier for mucosal starch digestion. Acarbose inhibition was most potent against maltase-glucoamylase activities of the wild-type mouse. The consortium of 6 interactive enzymes appears to be a mechanism for adaptation of alpha-glucogenesis to a wide range of food starches.

Morphogenesis of the peri-implant mucosa: an experimental study in dogs

PURPOSE: The objective of the present experiment was to study the morphogenesis of the mucosal attachment to implants made of c.p. titanium. MATERIAL AND METHODS: All mandibular premolars were extracted in 20 Labrador dogs. After a healing period of 3 months, four implants (ITI Dental Implant System) were placed in the right and left sides of the mandible. A non-submerged implant installation technique was used and the mucosal tissues were secured to the conical marginal portion of the implants with interrupted sutures. The sutures were removed after 2 weeks and a plaque control program including daily cleaning of the remaining teeth and the implants was initiated. The animals were sacrificed and biopsies were obtained at various intervals to provide healing periods extending from Day 0 (2 h) to 12 weeks. The mandibles were removed and placed in the fixative. The implant sites were dissected using a diamond saw and processed for histological analysis. RESULTS: Large numbers of neutrophils infiltrated and degraded the coagulum that occupied the compartment between the mucosa and the implant during the initial phase of healing. At 2 weeks after surgery, fibroblasts were the dominating cell population in the connective tissue interface but at 4 weeks the density of fibroblasts had decreased. Furthermore, the first signs of epithelial proliferation were observed in specimens representing 1-2 weeks of healing and a mature barrier epithelium occurred after 6-8 weeks of healing. The collagen fibers of the mucosa were organized after 4-6 weeks of healing. CONCLUSION: It is suggested that the soft-tissue attachment to implants placed using a non-submerged installation procedure is properly established after several weeks following surgery.

Host responses to intestinal microbial antigens in gluten-sensitive mice

BACKGROUND AND AIMS: Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice. METHODOLOGY/PRINCIPAL FINDINGS: HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-gamma in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota. CONCLUSION: Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-gamma production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.

Microbial-immune cross-talk and regulation of the immune system

We are all born germ-free. Following birth we enter into a lifelong relationship with microbes residing on our body's surfaces. The lower intestine is home to the highest microbial density in our body, which is also the highest microbial density known on Earth (up to 10(12) /g of luminal contents). With our indigenous microbial cells outnumbering our human cells by an order of magnitude our body is more microbial than human. Numerous immune adaptations confine these microbes within the mucosa, enabling most of us to live in peaceful homeostasis with our intestinal symbionts. Intestinal epithelial cells not only form a physical barrier between the bacteria-laden lumen and the rest of the body but also function as multi-tasking immune cells that sense the prevailing microbial (apical) and immune (basolateral) milieus, instruct the underlying immune cells, and adapt functionally. In the constant effort to ensure intestinal homeostasis, the immune system becomes educated to respond appropriately and in turn immune status can shape the microbial consortia. Here we review how the dynamic immune-microbial dialogue underlies maturation and regulation of the immune system and discuss recent findings on the impact of diet on both microbial ecology and immune function.

Exposure to food allergens through inflamed skin promotes intestinal food allergy through the thymic stromal lymphopoietin-basophil axis.

BACKGROUND Exposure to food allergens through a disrupted skin barrier has been recognized as a potential factor in the increasing prevalence of food allergy. OBJECTIVE We sought to test the immunologic mechanisms by which epicutaneous sensitization to food allergens predisposes to intestinal food allergy. METHODS Mice were epicutaneously sensitized with ovalbumin or peanut on an atopic dermatitis-like skin lesion, followed by intragastric antigen challenge. Antigen-specific serum IgE levels and T(H)2 cytokine responses were measured by ELISA. Expression of type 2 cytokines and mast cell proteases in the intestine were measured by using real-time PCR. Accumulation of basophils in the skin and mast cells in the intestine was examined by using flow cytometry. In vivo basophil depletion was achieved by using diphtheria toxin treatment of Baso-DTR mice. For cell-transfer studies, the basophil population was expanded in vivo by means of hydrodynamic tail vein injection of thymic stromal lymphopoietin (TSLP) cDNA plasmid. RESULTS Sensitization to food allergens through an atopic dermatitis-like skin lesion is associated with an expansion of TSLP-elicited basophils in the skin that promote antigen-specific T(H)2 cytokine responses, increased antigen-specific serum IgE levels, and accumulation of mast cells in the intestine, promoting the development of intestinal food allergy. Critically, disruption of TSLP responses or depletion of basophils reduced the susceptibility to intestinal food allergy, whereas transfer of TSLP-elicited basophils into intact skin promoted disease. CONCLUSION Epicutaneous sensitization on a disrupted skin barrier is associated with accumulation of TSLP-elicited basophils, which are necessary and sufficient to promote antigen-induced intestinal food allergy.

IL-33 promotes an innate immune pathway of intestinal tissue protection dependent on amphiregulin-EGFR interactions

The barrier surfaces of the skin, lung, and intestine are constantly exposed to environmental stimuli that can result in inflammation and tissue damage. Interleukin (IL)-33-dependent group 2 innate lymphoid cells (ILC2s) are enriched at barrier surfaces and have been implicated in promoting inflammation; however, the mechanisms underlying the tissue-protective roles of IL-33 or ILC2s at surfaces such as the intestine remain poorly defined. Here we demonstrate that, following activation with IL-33, expression of the growth factor amphiregulin (AREG) is a dominant functional signature of gut-associated ILC2s. In the context of a murine model of intestinal damage and inflammation, the frequency and number of AREG-expressing ILC2s increases following intestinal injury and genetic disruption of the endogenous AREG-epidermal growth factor receptor (EGFR) pathway exacerbated disease. Administration of exogenous AREG limited intestinal inflammation and decreased disease severity in both lymphocyte-sufficient and lymphocyte-deficient mice, revealing a previously unrecognized innate immune mechanism of intestinal tissue protection. Furthermore, treatment with IL-33 or transfer of ILC2s ameliorated intestinal disease severity in an AREG-dependent manner. Collectively, these data reveal a critical feedback loop in which cytokine cues from damaged epithelia activate innate immune cells to express growth factors essential for ILC-dependent restoration of epithelial barrier function and maintenance of tissue homeostasis.

The IL-33/ST2 pathway contributes to intestinal tumorigenesis in humans and mice.

Colorectal cancer (CRC) develops through a multistep process and is modulated by inflammation. However, the inflammatory pathways that support intestinal tumors at different stages remain incompletely understood. Interleukin (IL)-33 signaling plays a role in intestinal inflammation, yet its contribution to the pathogenesis of CRC is unknown. Using immunohistochemistry on 713 resected human CRC specimens, we show here that IL-33 and its receptor ST2 are expressed in low-grade and early-stage human CRCs, and to a lesser extent in higher-grade and more advanced-stage tumors. In a mouse model of CRC, ST2-deficiency protects from tumor development. Moreover, bone marrow (BM) chimera studies indicate that engagement of the IL-33/ST2 pathway on both the radio-resistant and radio-sensitive compartment is essential for CRC development. Mechanistically, activation of IL-33/ST2 signaling compromises the integrity of the intestinal barrier and triggers the production of pro-tumorigenic IL-6 by immune cells. Together, this data reveals a tumor-promoting role of IL-33/ST2 signaling in CRC.

ADAPTATION OF INTESTINAL MUSCLE IN THE RAT AFTER INTESTINAL BYPASS

After intestinal bypass, the mucosa of the in-continuity segment (ICS) of intestine undergoes adaptive hyperplasia which results in increased absorptive function per length of intestine. In the present study, 70% of the small intestine was bypassed in rats to determine if intestinal muscle also adapts after bypass. To determine the effect of bypass on intestinal transit, a poorly absorbed marker substance was introduced into the orad portion of the ICS or bypassed loop (BL). Significantly less marker (P < 0.05) was passed from the ICS into the colon in 50 minutes in fed rats at 14 days compared to at 3 days after bypass. In 150 minutes there was more marker in the colon of fed rats at 3 and 14 days but not at 35 days after bypass than in control. In the BL, transit was slowed significantly in fed rats at 3 and 35 days and in fasted rats at 3 days but not 35 days after bypass compared to control. The circular muscle from the BL and the distal but not proximal portion of the ICS developed significantly more carbachol-stimulated force in vitro at 35 but not 3 days after bypass compared to unoperated but not sham-operated controls. At 35 days after bypass, the muscle layers had a greater muscle wet weight and protein content compared to both unoperated and sham-operated control in both the proximal and distal portions of the ICS. Similarly, there was more muscle in histological sections of the BL and distal portion of the ICS at 35 days after bypass compared to either control. Nonetheless, at 35 days after bypass actomyosin content as a fraction of muscle weight or total protein content was not different from control. The results support the hypothesis that there was a functional adaptation, i.e. slowed transit in fed rats that allowed more time for absorption. Feeding caused slowed transit in the BL as well as the ICS. Other results suggest that an increased amount of functional muscle formed in the distal portion of the ICS after bypass. ^

MODULATION OF HOST INTESTINAL MYOELECTRIC ACTIVITY BY LOCAL ANAPHYLAXIS: A COMPONENT OF PROTECTIVE IMMUNITY TO AN ENTERIC PARASITE (TRICHINELLA SPIRALIS, EIMERIA NIESCHULZI, BOWEL MOTILITY)

The hypothesis tested was that rapid rejection of Trichinella spiralis infective larvae from immunized rats following a challenge infection is associated with a local anaphylactic reaction, and this response should be reflected in altered small intestinal motility. The objective was to determine if altered gut smooth muscle function accompanies worm rejection based on the assumption that anaphylaxis in vivo could be detected by changes in intestinal smooth muscle contractile activity (ie. an equivalent of the Schultz-Dale reaction or in vitro anaphylaxis). The aims were to (1) characterize motility changes by monitoring intestinal myoelectric activity in conscious rats during the enteric phase of T. spiralis infection in immunized hosts, (2) detect the onset and magnitude of myoelectric changes caused by challenge infection in immunized rats, (3) determine the parasite stimulus causing changes, and (4) determine the specificity of host response to stimulation. Electrical slow wave frequency, spiking activity, normal interdigestive migrating myoelectric complexes and abnormal migrating action potential complexes were measured. Changes in myoelectric parameters induced by larvae inoculated into the duodenum of immune hosts differed from those associated with primary infection with respect to time of onset, magnitude and duration. Myoelectric changes elicited by live larvae could not be reproduced by inoculation of hosts with dead larvae, larval excretory-secretory products, or by challenge with a heterologous parasite, Eimeria nieschulzi. These results indicate that (1) local anaphylaxis is a component of the initial response to T. spiralis in immune hosts, since the rapid onset of altered smooth muscle function parallels in time the expression of rapid rejection of infective larvae, and (2) an active mucosal penetration attempt by the worm is necessary to elicit this host response. These findings provide evidence that worm rejection is a consequence of, or sequel to, an immediate hypersensitivity reaction elicited when parasites attempt to invade the gut mucosa of immunized hosts. ^

Relation between acid back -diffusion and luminal surface hydrophobicity in canine gastric mucosa: Effects of salicylate and prostaglandin

The stomach is thought to be protected from luminal acid by a gastric mucosal barrier that restricts the diffusion of acid into tissue. This study tested the hypothesis that the hydrophobic luminal surface of canine gastric mucosa incubated in Ussing chambers, impedes the back-diffusion of luminal acid into the tissue. Isolated sheets of mucosa were treated with cimetidine to inhibit spontaneous acid secretion, and incubated under conditions that prevented significant secretion of luminal bicarbonate. By measuring acid loss from the luminal compartment using the pH-stat technique, acid back-diffusion was continuously monitored; potential difference (PD) was measured as an index of tissue viability. Tissue luminal surface hydrophobicity was estimated by contact angle analysis at the end of each experiment. Addition of 16,16-dimethyl prostaglandin E$\sb2$ to the nutrient compartment enhanced luminal surface hydrophobicity, but did not reduce acid back-diffusion in tissues that maintained a constant PD. 10 mM salicylate at pH 4.00 in the luminal compartment reduced surface hydrophobicity, but this decrease did not occur if 1 ug/ml prostaglandin was present in the nutrient solution. Despite possessing relatively hydrophilic and relatively hydrophobic surface properties, respectively, acid back-diffusion in the absence of salicylate was not significantly different between these two groups. Neither group maintained a PD after incubation with salicylate. Lastly, radiolabelled salicylate was used to calculate the free (non-salicylate associated) acid loss in tissues incubated with salicylate and/or prostaglandin. No significant correlation was found between free acid back-diffusion and luminal surface hydrophobicity. These data do not support the hypothesis that acid back-diffusion in impeded by the hydrophobic surface presented by isolated canine gastric mucosa. ^